Over the past decade it has become apparent that DNA in the human genome has considerable potential to form structures other than canonical Watson-Crick duplexes. Among the motifs most prone to form structures in vitro are runs of guanines, which readily assemble into structures stabilized by G quartets. G quartets can stabilize a variety of distinct structures, which we refer to collectively as G4 DNA. Our laboratory has identified and characterized activities that recognize G4 DNA with high affinity and specificity and dynamically alter its structure. The affinity and specificity of these factors for G4 DNA substrates argue that G4 DNA forms in vivo, and that it is the target for specific factors involved in DNA metabolism. We now propose (1) to ask directly if G4 DNA forms in vivo; (2) to learn how G4 DNA is recognized by RecQ family helicases; (3) to study how G4 DNA causes or contributes to genomic instability associated with cancer predisposition; (4) to study cleavage of G4 DNA in mammalian cells; (5) and to ask whether repair pathways eliminate G4 DNA. The proposed experiments will provide detailed mechanistic understanding of how DNA sequence impacts chromosome replication and genomic structure. They will elucidate mechanisms of maintenance of critical regions of the human genome, the rDNA and telomeres. They will also have immediate impact on our understanding of human health and disease, because the failure to remove G4 DNA may be associated with genetic instability that leads to malignancy.